Apoptosis In Human Osteosarcoma Cells Research Articles

Effects of the Long Non-Coding RNA HOST2 On the Proliferation, Migration, Invasion and Apoptosis of Human Osteosarcoma Cells

This study aimed to explore the effects of the long non-coding RNA HOST2 (lnc-HOST2) on the proliferation, migration, invasion and apoptosis of osteosarcoma cells. Osteosarcoma tissues and adjacent normal tissues from 52 patients were selected. Human osteosarcoma cell lines (SaOS2, HOS, U2OS and MG-63) were collected and cultured; MG-63 cells had the highest lnc-HOST2 expression and thus were used in subsequent experiments. Then, MG-63 cells were transfected and divided into the blank (no transfection), si-CON (transfected with negative control siRNA) and si-lnc-HOST2 (transfected with small interference lnc-HOST2 siRNA) groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression of lnc-HOST2 in primary tissues and cells. Cell growth was detected using the CCK-8 and colony formation assays. Cell doubling time was detected. Cell migration and invasion were observed using the scratch test and Transwell assays. Cell apoptosis and cell cycle progression of osteosarcoma cells were detected using flow cytometry with annexin V/PI double staining and PI staining, respectively. The level of lnc-HOST2 expression in the si-lnc-HOST2 group was significantly decreased compared to that in the blank and si-CON groups. The OD values in the si-lnc-HOST2 group were significantly lower than those in the blank and si-CON groups. Compared to the blank and si-CON groups, the si-lnc-HOST2 group presented significant decreases in the colony number and healing rates after scratching. The number of invasive cells in the si-lnc-HOST2 group was significantly less than that in the blank and si-CON groups. In the si-lnc-HOST2 group, the cell cycle was mainly halted in the G1 phase, and the apoptosis rate and doubling time in this group were significantly higher than those in the blank group and si-CON group. Inhibition of lnc-HOST2 could suppress the proliferation, migration, and invasion and promote the apoptosis of osteosarcoma cells.

Zebularine enhances apoptosis of human osteosarcoma cells by suppressing methylation of ARHI.

ARHI is an imprinted tumor suppressor gene and its methylation suppresses ARHI transcription levels to cause the development and progression of malignant tumors. Zebularine exerts a demethylation function for tumor suppressor genes. Our study aims to investigate the effect and mechanism of action of zebularine on the epigenetic modification of the ARHI gene, and whether this effect may modulate the viability and apoptosis of human osteosarcoma cells. We found that zebularine inhibited the viability and promoted apoptosis in osteosarcoma cells. Zebularine potentiated the expression of ARHI at both the protein and mRNA level. This was related to the downregulation of methylation of ARHI caused by zebularine. Zebularine suppressed the interaction of DNA methyltransferase 1 (DNMT1) with histone methyltransferase G9a, but had no effect on G9a alone. Knockdown of DNMT1 or G9a can induce a reduction of ARHI methylation. Therefore, we inferred that zebularine was likely to directly repress DNMT1 alone, but G9a was necessary to regulate the function of DNMT1 on ARHI methylation. Moreover, knockdown of ARHI rescued cell viability and apoptosis under the zebularine‐treated condition. We showed that zebularine inhibited viability and promoted apoptosis by disturbing the interaction between DNMT1 and G9a, thereby resulting in lower ARHI methylation and elevated ARHI expression in osteosarcoma cells.

Combined Use of Zoledronic Acid Augments Ursolic Acid-Induced Apoptosis in Human Osteosarcoma Cells through Enhanced Oxidative Stress and Autophagy.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, triggers apoptosis in several tumor cell lines but not in human bone cancer cells. Most recently, we have demonstrated that UA exposure reduces the viability of human osteosarcoma MG-63 cells through enhanced oxidative stress and apoptosis. Interestingly, an inhibitor of osteoclast-mediated bone resorption, zoledronic acid (ZOL), also a third-generation nitrogen-containing bisphosphonate, is effective in the treatment of bone metastases in patients with various solid tumors. In this present study, we found that UA combined with ZOL to significantly suppress cell viability, colony formation, and induce apoptosis in two lines of human osteosarcoma cells. The pre-treatment of the antioxidant had reversed the oxidative stress and cell viability inhibition in the combined treatment, indicating that oxidative stress is important in the combined anti-tumor effects. Moreover, we demonstrated that ZOL combined with UA significantly induced autophagy and co-administration of autophagy inhibitor reduces the growth inhibitory effect of combined treatment. Collectively, these data shed light on the pathways involved in the combined effects of ZOL and UA that might serve as a potential therapy against osteosarcoma.

The Fractionated Toona sinensis Leaf Extract Induces Apoptosis of Human Osteosarcoma Cells and Inhibits Tumor Growth in a Murine Xenograft Model.

Background: Osteosarcoma is a malignant bone tumor prevalent in adolescents with poor prognosis. Toona sinensis showed potent antiproliferation effect on lung, melatonin, ovary, colon, and liver cancers. However, the effects of the species on osteosarcoma cells are rarely investigated. Results: In this study, we found fraction 1 of Toona sinensis leaf (TSL-1) resulted in inhibition of cell viability in MG-63, Saos-2, and U2OS osteosarcoma cell lines, while it only caused a moderate suppressive effect on normal osteoblasts. In addition, TSL-1 significantly elevated lactate dehydrogenase leakage and induced apoptosis and necrosis in Saos-2 cells. TSL-1 increased mRNA expression of pro-apoptotic factor Bad. Most important, TSL-1 significantly suppressed Saos-2 xenograft tumor growth in nude mice by increasing caspase-3. The IC-50 of TSL-1 for the 3 tested osteosarcoma cells is around 1/9 of that for lung cancer cells. Conclusion: We demonstrated that TSL-1, a fractionated extract from TSL, caused significant cytotoxicity to osteosarcoma cells due to apoptosis. In vivo xenograft study showed that TSL-1 suppressed the growth of osteosarcoma cells at least in part by inducing apoptosis. Our results indicate that TSL-1 has potential to be a promising anti-osteosarcoma adjuvant functional plant extract.

AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway.

The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma.

Interferon α sensitizes human osteosarcoma cells to doxorubicin-induced apoptosis through p53-dependent pathway

Objective: To investigate the effect of IFNα on doxorubicin-induced apoptosis in human osteosarcoma cells with its molecular mechanisms to provide evidences for improving the treatment of osteosarcoma. Methods: Osteosacoma U2OS and MG63 cells were treated with IFNα and Doxorubicin, alone or in combination, for 72 h . Cytotoxicity was determined with MTT. Apoptosis was evaluated through fluorescence-activated cell sorting, Hoechst33258 staining and DNA ladder assay. The expression of p53, Bax, Bcl-2, Mdm2, p21, caspase-3 and PARP was determined with Western blot. siRNA interference was used to silence p53. Results: IFNα treatment for 72 h did not induce cytotoxicity but greatly enhanced doxorubicin-induced cytotoxicity and apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells. Compared with other groups, the combination of IFNα and doxorubicin induced more obvious apoptotic morphological changes and DNA ladder. IFNα did not alter the expression of the indicate genes. The expression of p53, Bax, Mdm2 and p21 was up-regulated by doxorubicin and further increased in response to combination. The expression of Bcl-2 was down-regulated by doxorubicin and further decreased in response to combination.There were no differences among groups in MG63 cells. The expression of p53 was effectively blocked by p53-siRNA in U2OS cells. The p53 silencing greatly reduced the cytotoxicity mediated by combination for 72 h, compared with non- and control-siRNA groups. The activation of caspase-3 and PARP mediated by combination was largely suppressed by p53 silence. Conclusion: IFNα sensitizes human osteosarcoma cells to doxorubicin-induced apoptosis through p53-dependent pathway. The combination of IFNα and traditional chemotherapy can be used in osteosarcoma treatment.

Celastrol negatively regulates cell invasion and migration ability of human osteosarcoma via downregulation of the PI3K/Akt/NF-κB signaling pathway in vitro.

Osteosarcoma (OS) is a primary malignant tumor of the bone, with a tendency to metastasize early. Despite the advances in treatment options, more than 30% of patients develop distant metastases, and the prognosis of these patients with metastases is extremely poor. Celastrol has been demonstrated to manifest multiple pharmacological activities, including induction of apoptosis in numerous types of cancer cell lines. Our previous studies have also suggested that Celastrol is capable of inducing apoptosis of human osteosarcoma cells via the mitochondrial-dependent pathway. The purpose of this study was to investigate the effects of Celastrol on the migration and invasion of human osteosarcoma U-2OS cells in vitro. Cell migration and invasion were investigated using wound healing and Boyden chamber Transwell assays. We observed that Celastrol suppressed cell invasion and migration in human osteosarcoma U-2OS cells. Furthermore, protein expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K), Akt, inhibitor of κB kinase α/β, inhibitor of κB α, nuclear factor-κB (NF-κB subunit p65) and matrix metalloproteinase (MMP)-2 and -9 were measured by western blot analysis. We observed that the PI3K/Akt/NF-κB signaling pathway was inhibited following Celastrol treatment. In addition, the expression levels of MMP-2 and -9 proteins were also reduced significantly following Celastrol treatment. Therefore, we confirmed that Celastrol suppressed osteosarcoma U-2OS cell metastasis via downregulation of the PI3K/Akt/NF-κB signaling pathway in vitro.

Ursolic Acid Triggers Apoptosis in Human Osteosarcoma Cells via Caspase Activation and the ERK1/2 MAPK Pathway.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, has been shown to trigger apoptosis in several lines of tumor cells in vitro. We found that treatment with UA suppressed the viability of human osteosarcoma MG-63 cells and induced cell cycle arrest at sub-G1 and G2/M phases. Furthermore, exposure to UA induced intracellular oxidative stress and collapse of mitochondrial membrane permeability, resulting in the subsequent activation of apoptotic caspases 8, 9, and 3 as well as PARP cleavage, and ultimately apoptosis in MG-63 cells. Moreover, protein analysis of mitogen-activated protein kinase (MAPK)-related protein expression showed an increase in activated ERK1/2, JNK, and p38 MAPK in UA-treated MG-63 cells. In addition, UA-induced apoptosis was significantly abolished in MG-63 cells that had been pretreated with inhibitors of caspase 3, 8, and 9 and ERK1/2. Furthermore, UA-treated MG-63 cells also exhibited an enhancement in Bax/Bcl-2 ratio, whereas anti-apoptotic XIAP and survivin were down-regulated. Taken together, we provide evidence demonstrating that UA mediates caspase-dependent and ERK1/2 MAPK-associated apoptosis in osteosarcoma MG-63 cells.

MicroRNA-138 functions as a tumor suppressor in osteosarcoma by targeting differentiated embryonic chondrocyte gene 2.

BackgroundMicroRNA-138 (miR-138) has been proven to be a tumor suppressor gene in various types of tumors. However, the expression and the role of miR-138 in human osteosarcoma are still poorly understood. We investigated the function and the underlying mechanism of miR-138 in osteosarcoma.MethodsThe expression of miR-138 in human osteosarcoma tissues and cell lines was detected by real-time PCR analysis. The gain-of-function and loss-of-function experiments were performed on osteosarcoma cell lines to investigate the effects of miR-138 on osteosarcoma progression, and to determine whether differentiated embryonic chondrocyte gene 2 (DEC2) mediates these effects. Cell proliferation, apoptosis and invasion were assessed by MTT, flow cytometry and transwell-matrigel assays. Dual-luciferase reporter assay was used to identify whether DEC2 is a direct target of miR-138.ResultsMiR-138 was significantly downregulated in human osteosarcoma tissues and cell lines. Moreover, miR-138 expression was significantly lower in metastatic osteosarcoma tissues than that in non-metastatic tissues. The in vitro gain-of-function and loss-of-function experiments demonstrated that miR-138 inhibited cell proliferation and invasion, and promoted cell apoptosis of human osteosarcoma cells. DEC2 was verified as a direct target of miR-138, and DEC2 could reverse the inhibitory effect of miR-138 on osteosarcoma progression.ConclusionsThese findings suggested that miR-138 acts as a tumor suppressor in osteosarcoma.miR-138 inhibited cell proliferation and invasion, as well as promoted cell apoptosis of human osteosarcoma cells, at least partially, by inhibiting the expression of DEC2. MiR-138/DEC2 may be a novel therapeutic target in osteosarcoma.

GSK-3 inhibitor inhibits cell proliferation and induces apoptosis in human osteosarcoma cells.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that functions in numerous signaling pathways initiated by diverse stimuli. The functions of GSK-3 in cancer differ depending on cell type. In the present study, we examined the effects of a specific GSK-3 inhibitor on the regulation of osteosarcoma cell proliferation and apoptosis. Immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed to determine the expression pattern of GSK-3 in human osteosarcoma cells. We used the MTS assay, western blotting, measurement of single-stranded DNA and morphological analyses to study the effects of a GSK-3 inhibitor, SB216763 on osteosarcoma cell proliferation and survival. We detected an increase in mRNA expression of GSK-3 and aberrant nuclear accumulation of GSK-3 in the osteosarcoma cells. Pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of osteosarcoma cells. Inhibition of GSK-3 resulted in a decreased expression of Bcl-2 and a subsequent increase in osteosarcoma cell apoptosis via the mitochondrial pathway. The present study demonstrated that GSK-3 activity is critical for tumorigenicity and cell survival in osteosarcoma cells. Our findings suggest that GSK-3 is a potential therapeutic target for the treatment of human osteosarcoma.

Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway.

Recently, the human ether à go-go (eag) related gene 1 (hERG1) channel, a member of the voltage-dependent potassium channel (Kv) family, was determined to have a critical role in cancer cell proliferation, invasion, tumorigenesis and apoptosis. However, the expression levels and functions of hERG1 in osteosarcoma cells remain poorly characterized. In this study, hERG1 transcript and protein levels in osteosarcoma cells and tissues were measured using semi-quantitative real time PCR (RT-PCR), Western blot, and immunohistochemistry. The effects of hERG1 knockdown on osteosarcoma cell proliferation, apoptosis and invasion were examined using CCK-8, colony formation, flow cytometry, caspase-3 activity, wound healing and transwell based assays. Furthermore, semi-quantitative RT-PCR, Western blot and a luciferase reporter assay were used to assess the effects of hERG1 inhibition on the nuclear factor-κB (NF-κB) pathway. In addition, the effect of NF-κB p65-siRNA and NF-κB p65 expression on the survival of osteosarcoma cells was investigated. Through this work, a relationship for hERG1 with the NF-κB pathway was identified. Osteosarcoma cells and tissues were found to express high levels of hERG1. Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB. Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling. Taken together, these results demonstrate that hERG1 is necessary for regulation of osteosarcoma cellular proliferation, apoptosis and migration. Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

Dodecyl gallate induces apoptosis by upregulating the caspase-dependent apoptotic pathway and inhibiting the expression of anti-apoptotic Bcl-2 family proteins in human osteosarcoma cells.

Dodecyl gallate (DG) is a gallic acid ester that has been shown to inhibit tumor growth. The aim of this study was to investigate the mechanism by which DG induces antiproliferative and apoptotic effects in MG-63 human osteosarcoma cells. Dose- and time-dependent cytotoxic effects of DG were determined using an MTT assay. The results showed that the half-maximal inhibitory concentration (IC50) of DG in MG-63 cells was 31.15 µM at 24 h, 10.66 µM at 48 h, and 9.06 µM at 72 h. Flow cytometric analysis demonstrated that exposure to 20 and 40 µM DG resulted in an increase in the sub-G1 phase population and in S-phase cell cycle arrest. Furthermore, western blot analysis of apoptosis-related protein expression revealed an increase in the activation of caspases 8 and 3, cleavage of poly (ADPribose) polymerase (PARP), and disruption of mitochondrial membrane permeability was measured by flow cytometry. An increase in the Bax/Bcl-2 ratio and a decrease in the expression of inhibitor of apoptosis protein (IAP) family members, namely X-linked inhibitor of apoptosis protein and survivin, were also observed following DG treatment. These data provide insight into the molecular mechanisms governing the ability of DG to induce apoptosis in human osteosarcoma cells in vitro.

A novel schiff base zinc coordination compound inhibits proliferation and induces apoptosis of human osteosarcoma cells.

Various kinds of schiff base metal complexes have been proven to induce apoptosis of tumor cells. However, it remains largely unknown whether schiff base zinc complexes induce apoptosis in human cancer cells. Here, we synthesized a novel schiff base zinc coordination compound (SBZCC) and investigated its effects on the growth, proliferation and apoptosis of human osteosarcoma MG-63 cells. A novel SBZCC was synthesized by chemical processes and used to treat MG-63 cells. The cell viability was determined by CCK-8 assay. The cell cycle progression, mitochondrial membrane potential and apoptotic cells were analyzed by flow cytometry. The apoptosis-related proteins levels were determined by immunoblotting. Treatment of MG-63 cells with SBZCC resulted in inhibition of cell proliferation and cell cycle arrest at G1 phase. Moreover, SBZCC significantly reduced the mitochondrial membrane potential and induced apoptosis, accompanied with increased Bax/Bcl-2 and FlasL/Fas expression as well as caspase-3/8/9 cleavage. Our results demonstrated that the synthesized novel SBZCC could inhibit the proliferation and induce apoptosis of MG-63 cells via activating both the mitochondrial and cell death receptor apoptosis pathways, suggesting that SBZCC is a promising agent for the development as anticancer drugs.

P14ARF enhances cisplatin-induced apoptosis in human osteosarcoma cells in p53-independent pathway

To study effect of tumor suppressor p14ARF on cisplatin-induced apoptosis in human osteosarcoma cells with its molecular mechanisms to provide evidences for increasing chemosensitivity of osteosarcoma. pcDNA3.1-p14ARF plasmid was stable transfected into MG63 cells lack of p14ARF expression. Expression of p14ARF on mRNA and protein level was evaluated with RT-PCR and Western blot. MG63, MG63-vec and MG63-ARF cells were treated with cisplatin. Cell growth inhibition and IC50 were determined through MTT assay. Apoptosis was detected using fluorescence-activated cell sorting and Hoechst33258 staining. The expression of p53, Bax, p21, Mdm2, Fas, Caspase-3, caspase-9 and PARP was detected with Western blot. RNAi was used to silence p53. Cells were pre-treated with Caspase-9 specific inhibitor Z-LEHD-FMK to determine whether the effect was Caspase-9-dependent. There was no expression of p14ARF in MG63 and MG63-vec cells but obvious expression in MG63-ARF cells on mRNA and protein level. Cell viability was 84.2%±4.3%, 80.8%±4.3% and 58.9%±5.4% in MG63, MG63-vec, and MG63-ARF cells after treatment of cisplatin for 72 h. IC50 was (11.1±0.6), (10.7±0.9) and (7.2±0.7) µmol/L. The apoptotic rate was 13.6%, 18.5% and 35.9% in groups, There were more obvious apoptotic more changes in MG63-ARF cells than MG63 and MG63-vec cells, and activation of Caspase-3, 9 and PARP on higher level in U2OS-ARF cells after stimulation with cisplatin for 72 h. The expression of p53, Bax, p21, Mdm2 and Fas, in MG63-vec and MG63-ARF cells did not changed (P>0.05). The expression of p53 was effectively and continuously suppressed by p53-siRNA in U2OS-vec and U2OS-ARF cells. The p53 silencing did not alter the cytotoxicity mediated by cisplatin treatment for 72 h (P>0.05). Cell viability was 96.8%±3.6%, 54.1%±5.7% and 89.5%±5.1% in Z-LEHD-FMK, cisplatin and Z-LEHD-FMK+cisplatin groups. p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma MG63 cells in p53-independent caspase-9-dependent pathway, in which the intrinsic mitochondrial apoptotic pathway is involved.

Celastrol induces apoptosis of human osteosarcoma cells via the mitochondrial apoptotic pathway.

Celastrol is an active compound extracted from the root bark of Triptergium wilfordii Hook F., also known as 'Thunder of God Vine'. It is a well-known Chinese medicinal herb that was found to inhibit tumor cell growth and promote apoptosis in several tumor cell lines. However, research into its effects on osteosarcoma cell apoptosis is still extremely limited. The present study was undertaken to determine the effect of celastrol on viability and apoptosis of osteosarcoma cells and furthermore, to illuminate the molecular mechanism of celastrol-induced osteosarcoma cell apoptosis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to evaluate the viability of the cells following treatment with celastrol. The effect of celastrol on the apoptotic rate of the cells was evaluated by flow cytometry using Annexin V-PE/7-AAD staining assay. Fluorescence microscopy was used to detect the morphological changes in the human osteosarcoma U-2OS cell lines. The expression of Bcl-2 family proteins, caspase-3, caspase-8, caspase-9, cytochrome c and PARP was measured by western blotting. We found that celastrol significantly inhibited the growth of osteosarcoma cells in a dose-dependent manner, particularly U-2OS cells. Furthermore, we observed that celastrol upregulated the expression of the pro-apoptotic proteins Bax and cytochrome c and altered the ratio of Bax/Bcl-2, and triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and -9 activation and PARP cleavage. To conclude, the results indicate that celastrol inhibits the proliferation of human osteosarcoma cancer cells by inducing apoptosis via the mitochondrial-dependent pathway.

Molecular mechanisms of luteolin induced growth inhibition and apoptosis of human osteosarcoma cells.

Luteolin is a flavone in medicinal plants as well as some vegetables and spices. It is a natural anti-oxidant with less pro-oxidant potential but apparently with a better safety profile. The purpose of this study was to investigate the molecular mechanism of luteolin-mediated apoptosis of MG-63 human osteosarcoma cells. MTT assay kit was employed to evaluate the effects of luteolin on MG-63 cells proliferation. Then, we performed Annexin V-FITC/PI to analyze the apoptotic rate of the cells. Furthermore, the inhibitory effects of luteolin on the expressions of BCL-2, BAX, Caspase-3 and Survivin were detected by Western blotting. As expected, luteolin (0.5, 2.5, 12.5 µg/mL) inhibited the growth of MG-63 cells by inhibiting cell proliferation and inducing cell apoptosis. Western blotting demonstrated that luteolin (0.5, 2.5, 12.5 µg/mL) inhibited the expressions of BCL-2, Caspase-3 and Survivin, and promoted the expression of BAX in MG-63 cells with a concentration dependent way. Luteolin can inhibit osteosarcoma cell proliferation and induce apoptosis effectively in a dose dependent manner through down-regulating the expression of BCL-2, Caspase-3 and Survivin proteins levels and up-regulating the expression of BAX protein level. These findings indicated that luteolin may be used as a novel herbal medicine for the treatment of osteosarcoma.

Delivery of inhibitor of growth 4 (ING4) gene significantly inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cells.

Growing evidence has suggested that inhibitor of growth 4 (ING4), a novel member of ING family proteins, plays a critical role in the development and progression of different tumors via multiple pathways. However, the function of ING4 in human osteosarcoma remains unclear. To understand its potential roles and mechanisms in inhibiting osteosarcoma, we constructed an expression vector pEGFP-ING4 and transfected the human osteosarcoma cells using this vector. We then studied the effects of over-expressed ING4 in the transfected cells on the proliferation, apoptosis and invasion of the osteosarcoma cells. The up-regulation of ING4 in the osteosarcoma cells, arising from the stable pEGFP-ING4 gene transfection, was found to significantly inhibit the cell proliferation by the cell cycle alteration with S phase reduction and G0/G1 phase arrest, induce cell apoptosis via the activation of the mitochondria pathway, and suppress cell invasion through the down-regulation of the matrix metalloproteinase 2 (MMP-2) and MMP-9 expression. In addition, increased ING4 level evoked the blockade of NF-κB signaling pathway and down-regulation of its target proteins. Our work suggests that ING4 can suppress osteosarcoma progression through signaling pathways such as mitochondria pathway and NF-κB signaling pathway and ING4 gene therapy is a promising approach to treating osteosarcoma.

Effect of microRNA-101 on proliferation and apoptosis of human osteosarcoma cells by targeting mTOR.

Studies have proved that microRNA-101 (miR-101) functions as a tumor suppressor and is associated with growth and apoptosis of various human cancers. However, the role of miR-101 in osteosarcoma and the possible mechanism by which miR-101 affects the tumor growth and apoptosis have not been fully elucidated. In this study, we found that the expression of miR-101 was down-regulated in osteosarcoma tissues and Saos-2 cell line as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. To better characterize the role of miR-101 in osteosarcoma, we used a gain-of-function analysis by transfecting human osteosarcoma cell line Saos-2 with chemically synthesized miR-101 mimics. The results showed that overexpression of miR-101 inhibited the proliferation and promoted the apoptosis of Saos-2 cells. Meanwhile, bioinformatic analysis demonstrated that mTOR gene was a direct target of miR-101. Overexpression of miR-101 significantly decreased the expression of mTOR at both mRNA and protein levels in Saos-2 cells, consequently inhibiting Saos-2 cells proliferation and promoting cells apoptosis in an mTOR-dependent manner. Taken together, these data suggest that miR-101 may act as a tumor suppressor, which is commonly downregulated in both osteosarcoma tissues and cells. mTOR plays an important role in mediating miR-101 dependent biological functions in osteosarcoma. Reintroduction of miR-101 may be a novel therapeutic strategy by down-regulating mTOR expression.

Epigenetic silencing of methyl-CpG-binding protein 2 gene affects proliferation, invasion, migration, and apoptosis of human osteosarcoma cells.

Methyl-CpG-binding protein 2 (MeCP2) is a DNA methylation-related gene of the methyl-CpG-binding protein family. Here, we investigated the epigenetic function of the MeCP2 in SaOS2 and U2OS cell lines, and explored the antitumor effects of the gene silencing for osteosarcoma. In this study, chromatin immunoprecipitation assay was used to detect MeCP2 binding activity with TSSC3 gene. RT-PCR and western blot assay were used to analyze the MeCP2 expression in osteosarcoma cell lines after transfection with LV-MECP2-RNAi. Transwell invasion and migration assays were used to detect the cell invasion and migration. The cell apoptosis was examined by using the flow cytometry assay. The tumor size was also assessed to determine the therapeutic effects of gene silencing. The results indicated that MeCP2 indicated the highest combining power with TSSC3 gene. LV-MECP2-RNAi could decrease MeCP2 level in tumor cells compared with the untreated cells (P < 0.05). LV-MECP2-RNAi inhibited the U2OS and SaOS2 cells invasion and migration compared with the control cells (P < 0.05). LV-MECP2-RNAi triggered the U2OS and SaOS2 cell apoptosis, and inhibited the cell proliferation significantly compared with the control cells (P < 0.05). The gene silencing of RNAi could also decreased the tumor size significantly compared with untreated cells (P < 0.05). In conclusion, silencing the MeCP2 gene could block the MeCP2 expression and inhibit the tumor cell migration, invasion, and proliferation, and decreases the tumor size by inducing the apoptosis of the tumor cells.

CUL4B promotes proliferation and inhibits apoptosis of human osteosarcoma cells.

Cullin 4B (CUL4B) is a component of the Cullin4B-Ring E3 ligase complex (CRL4B) that functions in proteolysis and is implicated in tumorigenesis. Here, we report that CUL4B is associated with tumorigenesis by promoting proliferation and inhibiting apoptosis of human osteosarcoma cells. We performed RNA interference (RNAi) with a lentiviral vector system to silence the CUL4B gene using osteosarcoma SAOS-2 cells. The negative control included the normal target cells infected with the negative control virus whereas the knockdown cells included the normal target cells transfected with the RNAi target virus. We assessed the inhibition resulting from the decreased expression of the CUL4B gene on the proliferation rate of SAOS-2 cells, and also evaluated the cell cycle distribution, apoptosis and clonability. Compared with the negative control, the CUL4B gene expression was significantly inhibited in the SAOS-2 cells at the mRNA and protein levels in the knockdown group (P<0.01). Furthermore, in the knockdown group, the cell proliferation rate and clonability were also significantly inhibited (P<0.01). The apoptosis rate increased significantly (P<0.05). A significant decrease in the number of cells in the G1 phase (P<0.01) and significant increases in the S (P<0.01) and G2 phases (P<0.05) were observed. The silencing of CUL4B gene expression can effectively inhibit osteosarcoma cell proliferation and induce apoptosis. These findings may provide a novel biomarker for the treatment of osteosarcoma.